地衣芽孢杆菌GXN151的纤维素酶基因cel12A的序列分析及其在大肠杆菌中的表达

地衣芽孢杆菌菌株GXNl51的cell2A基因为783bp,可编码含261个氨基酸的羧甲基纤维素酶Cel12A,Cel12A的N-末端第1～28氨基酸具典型的信号肽特征、第9～31氨基酸具跨膜功能域特征、第104～261氨基酸形成家族12糖基水解酶(glycosyl hydrolase)功能域。将编码其第73-261氨基酸的DNA序列克隆到大肠杆菌表达载体pET一30a( )得表达质粒pGXNl2A,用1mmol／IPTG诱导处理JMl09(DE3)／pGXNl2A的培养液6h pGXNl2A的表达量达到最高,在JMl09(DE3)和BL21(DE3)pLysS中该表达蛋白质可分别占菌体胞内总蛋白的54．3％和20．9％。在含羧甲基纤维素的LA平板上JMl09(DE3)／pGXNl2A和BL21(DE3)pLysS／pGXNl2A表现有较弱的羧甲基纤维素酶活性,说明重组的Cell2A的催化功能域具有独立的催化活性。包含体检测表明pGXNl2A在JMl09(DE3)中的表达产物大部分形成不溶性包含体。     

枯草芽孢杆菌B11基因文库的构建及与拮抗物质的生物合成相关基因的克隆

枯草芽孢杆菌菌株B 11对广泛的植物病原真菌和细菌都具有拮抗作用。以柯斯质粒pW EB∷TNC为载体构建了枯草芽孢杆菌菌株B 11的基因文库,文库含9 000个克隆。文库克隆中插入的DNA片段平均为42.1 kb,该文库含有菌株B 11基因组中任一基因的概率为99.99%。采用平板活性检测法筛选文库,筛选到1个对茄青枯假单胞菌菌株P 13具有拮抗活性的文库克隆GXN 9527,该克隆的重组质粒pGXN 9527含有50 kb的菌株B 11的DNA。文库克隆对革兰氏阴性植物病原细菌如水稻黄单胞菌水稻变种也具有拮抗活性,而对革兰氏阳性细菌如地衣芽孢杆菌和植物病原真菌如尖孢镰刀菌西瓜专化型、立枯丝核菌、水稻稻灰梨孢菌则没有拮抗活性。分别含有pGXN 9527的18、12、9、8 kb B amHⅠ片段的亚克隆对P 13均没有拮抗活性,说明编码该拮抗物质的生物合成基因很可能成簇存在。     

水稻白叶枯病菌GX1329基因组文库的构建及含编码TAL效应物基因的克隆的分离

由革兰氏阴性细菌水稻白叶枯病菌引起的水稻白叶枯病是亚洲、北美以及非洲部分地区最严重的水稻病害之一,水稻白叶枯病可使水稻减产高达50％以上。研究表明水稻白叶枯病菌的毒力主要依靠三型分泌系统所分泌的效应物。为了解水稻白叶枯病菌广西菌株GX1329中含有avrBs3／pthA家族基因的情况,本研究应用Alu I部分酶切其基因组DNA,构建了含有736个克隆的菌株GX1329的基因组文库。BamHI酶切分析随机挑取的15个文库克隆表明,克隆的外源DNA随机性良好,克隆的最小片段为27．7kb,最大为58．5kb,平均大小为39．9kb,文库克隆容量约为2．8×10^3Mb,该文库中包含基因组中任一个基因的概率为99．4％。利用来自水稻白叶枯病菌菲律宾菌株PX086的无毒基因avrXa10的第252位～第486位核苷酸序列作为探针,通过菌落原位杂交从GX1329基因组文库中筛选到37个含avrBs3／pthA家族基因的克隆。再通过Southern杂交分析,得到了17个独立克隆。这17个克隆中至少含有13个不同的avrBs3／pthA家族基因。这些基因在GX1329基因组中有的单独存在,有的两个或两个以上串联存在。本工作基本上明确了菌株GX1329基因组中avrBs3／pthA家族基因的数量,为进一步研究菌株GX1329中avrBs3／pthA家族基因的功能奠定了基础。     

结晶纤维素降解细菌的筛选、分离与鉴定

采用以结晶纤维素（Avicel）为唯一碳源的平板活性筛选法,从60份森林腐殖土、腐术样品中分离得到2株在pH5．0、37℃条件下高效降解结晶纤维素的细菌菌株GXN152和GXN153。以菌株的总DNA基因为模板,用细菌的16S rRNA基因的通用引物扩增得到菌株GXN152和GXN153的16S rRNA基因序列。测序分析表明菌株GXN152的16S rRNA基因序列和洋葱假单胞菌（Burkholderia cepacia）菌株CNR22的16S rRNA基因序列的同源性最高,具有98％的同源性,生理生化特性检测表明菌株GXN152具有洋葱假单胞菌的鉴别特征,将结晶纤维素降解菌GXN152鉴定为洋葱假单胞菌。菌株GXN153的16S rRNA基因序列和类芽孢杆菌（Paenibacillus favisporus）菌株GMP01的16S rRNA基因序列的同源性最高,具有99％的同源性,生理生化特性检测表明菌株GXN153具有类芽孢杆菌的鉴别特征,将纤维素降解菌GXN153鉴定为类芽孢杆菌。     

芽孢杆菌α-淀粉酶基因的克隆和测序

从土样中筛选获得一株α－淀粉酶产量的芽孢杆菌菌株,编号为ＸＤ９１３。通过克隆和测序,得到了一个２０９０ｂｐ的ＤＮＡ片段,内含一个编码５１４个氨基酸的基因,经同源性比较,发现该基因与已发表的解淀粉芽孢杆菌的α－淀粉酶基因的同源性高达９９．８％。     

甘薯茎线虫(Ditylenchus destructor)β-1,4内切葡聚糖酶基因(Dd-eng-1b)cDNA全长的克隆与序列分析

β-1,4内切葡聚糖酶是植物寄生线虫口针分泌的一类细胞壁降解酶,在植物线虫的侵染过程中具有重要的作用。本文以甘薯茎线虫为材料,用RT-PCR和RACE方法,获得了β-1,4-内切葡聚糖酶基因的cDNA全长,并将该基因命名为Dd-eng-1b (GenBank登录号为FJ430142)。此cDNA全长序列为1640bp,包括一个1443bp的完整ORF,编码一含481个氨基酸的蛋白,其理论分子量与等电点分别为50.89KD,pI为6.94。序列比对分析表明含有糖基水解酶的保守结构域,属于纤维素酶第五家族成员,N端具有19个氨基酸残基组成的信号肽,C端含有细菌式样的纤维素结合域(CBDⅡ)。cDNA与基因组DNA重叠分析表明,此基因包含4个内含子,长度分别为36bp、76bp、187bp和344bp,切割点符合5‘-GT...AG-3’的规律。系统进化树分析表明,该基因与细菌 Bacillus.subtilis和Erwina carotovora 分泌的纤维素酶属于同一支。     

甘薯茎线虫β-1,4内切葡聚糖酶基因(Dd-eng-1b)cDNA全长的克隆与序列分析

β-1,4内切葡聚糖酶是植物寄生线虫食道腺分泌的一类细胞壁降解酶,在植物线虫的侵染过程中具有重要的作用.以马铃薯腐烂茎线虫(Ditylenchus destructor)为材料,用RT-PCR和RACE方法,获得了β-1,4-内切葡聚糖酶基因的cDNA全长,并将该基因命名为Dd-eng-1b(GenBank登录号为FJ430142).此cDNA全长序列为1 640 bp,包括1个1 443 bp的完整ORF,编码1含481个氨基酸的蛋白,其理论分子量为50.89 kD,等电点pI为6.94.序列比对分析表明,含有糖基水解酶的保守结构域,属于纤维素酶第五家族成员,N端具有19个氨基酸残基组成的信号肽,C端含有细菌式样的纤维素结合域(CBD Ⅱ).cDNA与基因组DNA重叠分析表明,此基因包含4个内含子,长度分别为36、76、187和344bp,切割点符合5'-GT……AG-3'的规律.系统进化树分析表明,该基因与细菌Bacillus subtilis和Erwina carotovora分泌的纤维素酶属于同一支,推测该基因可能来源于细菌的水平基因转移.     

枯草芽孢杆菌群β-甘露聚糖酶基因克隆及同源性分析

本文对33株枯草芽孢杆菌群菌株进行β-甘露聚糖酶活性筛选,其中的32株具有β-甘露聚糖酶活性,只有1株无β-甘露聚糖酶活性。通过基因克隆测序的方法获得33株枯草芽孢杆菌群菌株β-甘露聚糖酶基因编码区全序列,对酶基因进行同源性分析并构建系统发育树;在β-甘露聚糖酶基因系统发育树中,33株枯草芽孢杆菌群菌株聚为3个分支,分别是枯草芽孢杆菌分支、地衣芽孢杆菌分支和解淀粉芽孢杆菌分支;枯草芽孢杆菌、地衣芽孢杆菌和解淀粉芽孢杆菌β-甘露聚糖酶基因种内同源性大于91％,而种间同源性为60％～69％。     

碱性土壤基因组DNA的分离纯化和基因文库的构建

直接从广西南宁市凤凰纸业排污沟碱性土壤样品中抽提和分离纯化混合基因组DNA，所获得DNA的产量为每克土壤样品10～30pg。采用限制性内切酶EcoR1酶处理后，构建了以pGEM-3Zf( )为载体的DNA部分文库。文库的容量为23650个转化子。外源片段DNA平均大小为3．2kb。建库效率为每克环境样品获得6000个左右的含1～15kb外源随机插入片段的克隆。通过DNA序列测定和同源性比较，对从文库随机词取的16个转化子序列进行分析，发现13个外源插入片段包含序列尚未确定的DNA片段。     

有机肥施加对红壤中反硝化细菌nirK基因多样性影响

为了解有机肥施用对于红壤中反硝化细菌群落结构的影响,设置了4个处理:CK(不施肥)、LM(低量有机肥)、ML(高量有机肥+石灰)和HM(高量有机肥)进行研究,并通过末端限制性片段长度多态性(T-RFLP)和克隆文库的DNA测序估计了nirK基因的多样性。结果表明:各处理分别挑选的288个阳性克隆子可分为78个类型,各处理nirK文库中的优势类群属于同一种类型,在CK处理中的占比为51%,在其他3个处理中的占比依次为33%、32%和27%。4个文库之间两两的相似性在37.50%~45.34%,系统进化树分析表明,51个OTUs测序结果中6个OTUs与苍白杆菌属的相似性最高,占测序总数的11.8%;其余45个OTUs属于未培养类型,占测序总数的88.2%。有机肥添加有助于提高nirK基因的多样性,并且出现了红壤原始环境中未出现的反硝化细菌类型。     

Biomass and turnover of bacteria in a forest soil and a peat

Short-term changes in numbers and biomass of bacteria, determined by direct counts, are descrived for a subarctic mire and for the humus and mineral soil layers of pine forest podsol. For biomass, monthly fluctuations for 15 months are presented. Almost as large fluctuations in bacterial numbers were found during 2 weeks as were found during the whole year. Precipitation resulted in increases in bacterial biomass even when the soil moisture content was non-limiting for bacterial growth, but these increases did not last for more than 1–2 days. Observed rapid declines in bacterial numbers were interpreted as the result of grazing by the microfuana. Changes in cell sizes and shapes after rainfall indicated that even under favourable growth conditions only 15–30% of the bacteria were active. The increases in bacterial biomass were used to calculate a minimum production. For the bacteria in the peat the production over 9 days was 40% of the mean standing crop value per day. The corresponding values for the bacteria of the humus and mineral layer over 13 days were 19 and 15%. The average generation times, estimated from increases in numbers, were 39 h for the peat bacteria and 66 and 55 h for the bacteria of the humus and mineral layers respectively. Based on the number of falls of rain a yearly bacterial production value of 210 g d.w.m^?2 was calculated for the forest site. This figure is discussed in relation to the yearly energy input through primary production.     

Structural characteristics of clay-dominated soils of a marsh and a palaeosol in a crossed diagram

Although the shrink‐swell phenomenon of clays has been thoroughly studied, the in situ relation of the shrinkage curve to the structure profile is rarely presented from the shrinkage limit to the liquid limit. We studied the consolidated structure of clay‐dominated (<2 μm) soils formed on ‘pseudo‐liquid’ marsh sediments in the ‘Marais de l’Ouest’ (France). The profiles were studied in a grassland field and in a sunflower field from unsaturated surface soils down to deeper, saturated, levels characterized by a very large water content (100% by weight). The consolidation states were quantified recording cone resistance (Qd) profiles using a dynamic penetrometer in successive seasons. These Qd profiles were compared with the associated wet density and gravimetric water‐content profiles. Two consolidation depths were evident, the surface soil and a 130‐cm deep palaeosol. The seasonal Qd profiles demonstrate the partial irreversibility of the consolidation peaks associated with the surface soil and with the palaeosol. The shrinkage properties were established through drying curves of undisturbed test samples. In the void ratio (e)‐water content (W) and water content (W)‐saturation index (Sr) diagrams, the profiles as a whole exhibit only one clay soil behaviour from their pseudo‐liquid to plastic to solid states. Each Qd profile is represented by a hyperbolic curve in the e/Qd diagram. Represented in a (e – W – Qd —Sr) crossed diagram, the vertical evolution of the successive profiles shows the soil structure behaviour from the initial pseudo‐liquid sediment to the consolidated soil. A simple cone resistance recording associated with gravimetric water‐content profiles, characterizes the evolution of structural layers of soils for the seasonal drying‐wetting cycles, for the over‐consolidation associated with the palaeosol, and also for the effect of ploughing.     

C and N dynamics of a range of biogas slurries as a function of application rate and soil texture: a laboratory experiment

Digestates vary in composition and studies regarding their impact on C and N dynamics in soils are scarce. The objective was to analyse the C and N dynamics of digestates originating from various substrates applied to a sandy Cambisol and a silty Anthrosol. In three laboratory experiments (4–6 weeks), the effects of digestate properties, N rate and water content were tested. Averaged over both soils, 21% of the C supplied was emitted as CO₂. Potential NH₃ emissions during the first week ranged between 6% and 12% of NH₄⁺ present in the digestates. The emission factors in the sandy Cambisol were on average 1.2 and 2 times higher for CO₂ and potential NH₃, respectively, compared to the silty Anthrosol. Similarly, net nitrogen mineralization in the sandy Cambisol was approximately twice the N mineralized in the silty Anthrosol. Net nitrification was not influenced by soil texture or different digestates, but increased with increasing application rates and had highest values at 75% of water holding capacity. Our results indicate that the type of substrate input for anaerobic digestion influences the properties of the digestate and therefore the dynamics of C and N. However, soil texture can affect these dynamics markedly.     

Calibration and Evaluation of a Mercury Model for a Western Stream and Constructed Wetland

Numerous studies have shown that Steamboat Creek in Nevada is highly contaminated with mercury, with aqueous mercury concentrations more than two orders of magnitude greater than nearby mountain streams. One objective of this study was to determine if the new Spreadsheet-based Ecological Risk Assessment for the Fate of Mercury (SERAFM) model could be calibrated to the concentrations of unfiltered and dissolved total mercury, and unfiltered and dissolved MeHg in the water column for a reach on SBC and a related constructed wetland mesocosm for different seasons and residence times. SERAFM is a new U.S. Environmental Protection Agency steady state, single segment, mass balance mercury model that has been applied to lakes, and this study also examined the model’s applicability for modeling an arid flowing water environment in different seasons. The average combined error between observed and model-estimated mercury concentrations was 12% and 17% for the reach and mesocosm, respectively. Some recommendations are proposed that may allow SERAFM to better model flowing systems.     

二氯喹啉酸·苄嘧磺隆复合污染降解菌的特性研究

A bacterial strain, designated as LS, was isolated from a contaminated soil and was found to be capable of utilizing quinclorac, bensulfuronmethyl, and a mixture of the two as carbon and energy sources for growth. Strain LS was identified as Ochrobactrum sp. based on its physiological-biochemical properties, 16S rDNA sequences, and phylogenetic analysis. The extent of degradation of quinclorac and bensulfuronmethyl at initial concentrations of 1.5 and 0.1 g L^-1 was 90% and 67%, respectively, as measured by high performance liquid chromatography （HPLC）. When a herbicide mixture of 0.34 g L^-1 quinclorac and 0.02 g L^-1 bensulfuronmethyl was applied as carbon sources, quinclorac and bensulfuronmethyl were degraded at 95.7% and 67.5%, respectively. It appears that quinclorac is utilized more easily in a mixture than in a single state. The optimal temperature for growth of strain LS was 37 ℃. Strain LS grew well at pH 6 to 9 and had the highest degradation level for quinclorac and bensulfuronmethyl at an initial pH of 7 and 8, respectively. Addition of 0.25 g L^-1 yeast extract could promote the growth and extent of degradation of quinclorac and bensulfuronmethyl by strain LS. Strain LS also showed the capability to degrade other aromatic compounds such as catechol, propisochlor, 4-chloro-2-methylphenoxyacetic acid sodium （MCPA-Na） and imazethapy. The isolate LS shows a huge potential to be used in bioremediation for treating complex herbicide residues.     

Isolation and characterization of a novel tannase from a metagenomic library

A novel gene (designated as tan410) encoding tannase was isolated from a cotton field metagenomic library by functional screening. Sequence analysis revealed that tan410 encoded a protein of 521 amino acids. SDS-PAGE and gel filtration chromatography analysis of purified tannase suggested that Tan410 was a monomeric enzyme with a molecular mass of 55 kDa. The optimum temperature and pH of Tan410 were 30 °C and 6.4. The activity was enhanced by addition of Ca(2+), Mg(2+) and Cd(2+). In addition, Tan410 was stable in the presence of 4 M NaCl. Chlorogenic acid, rosmarinic acid, ethyl ferulate, tannic acid, epicatechin gallate and epigallocathchin gallate were efficiently hydrolyzed by recombinant tannase. All of these excellent properties make Tan410 an interesting enzyme for biotechnological application.     

Compost and phosphorus amendments for stimulating microorganisms and growth of ryegrass in a Ferralsol and a Luvisol

A greenhouse pot experiment was carried out to investigate the effects of different P‐fertilizer application forms (triple superphosphate [TSP], compost + TSP, TSP‐enriched compost) on the growth of ryegrass and the soil microbial biomass. The fertilizers were applied at equivalent doses for all nutrients to a neutral Luvisol in comparison with an acidic Ferralsol. Fertilizer application led to significantly increased contents of microbial biomass C, N, and P. Furthermore, yields of shoot C and root C, and concentrations of P, Ca, Mg, K, Fe, and Mn in shoots and roots were significantly increased. These increases always followed the order TSP < compost + TSP < TSP‐enriched compost. Sole TSP application led only to maximum concentrations of N and S. In the Ferralsol, TSP had only minimal positive effect on the P concentration of the grass shoots. The positive effect of TSP‐enriched compost, i.e., incubating TSP together with compost for 24 h, did not differ between the neutral Luvisol and the acidic Ferralsol, i.e., the effect is independent of the soil type. Consequently, soluble inorganic P fertilizer should generally be mixed into an organic fertilizer before application to soil.     

某坝区滑坡变形破坏特征及其稳定性预判

雅砻江中游某巨型滑坡距离拟选坝址仅1.3 km,目前滑坡体上有裂缝发育,滑坡的整体稳定性对于坝址的选择具有颠覆性的影响。因此,在概要介绍滑坡地质特征基础上,对滑坡体上裂缝发育分布特征及其成因机理探讨,对裂缝的产生是由整体还是由于局部导致给出明确回答,进而依据滑坡工程地质特征及定量计算对蓄水后滑坡的整体稳定性作出预测。     

An analysis of food-web structure and function in a shortgrass prairie,a mountain meadow,and a lodgepole pine forest

Summary The structure of the below-ground detrital food web was similar in three different semiarid vegetation types: lodgepole pine (Pinus contorta subsp. latifolia), mountain meadow (Agropyron smithii), and shortgrass prairie (Bouteloua gracilis). The densities of component food-web functional groups and the response to removal of component groups, differed however. As measured by biomass, bacteria were dominant in the meadow and prairie, while fungi were dominant in the forest. Resourde-base dominance was reflected in consumer dominance, and both directly correlated with the form of inorganic N present. Bacterial-feeding nematodes were numerically dominant in the meadow and prairie, while microarthropods were dominant in the forest. Ammonium-N was the dominant form in the forest, while nitrate —nitrite-N was the more important form in both bacterial-dominated grasslands.Addition of a biocide solution containing carbofuran and dimethoate reduced the numbers of both microarthropods and nematodes. In the bacterial-dominated grasslands, these reductions resulted in no apparent effect on bacterial densities because one group of bacterial consumers (protozoa) increased following the decrease in bacteria-feeding nematodes, in increased fungal biomass, and in increased soil inorganic N. Conversely, in the forest, following the biocide-induced reduction in consumers, the total fungal biomass decreased, but inorganic-N levels increased. The meadow appeared to be the most resilient of the three ecosystems to biocide disturbance, as both nematode and arthropod numbers returned to control levels more rapidly in the meadow than in the prairie or the forest.